Optical methods for detecting particles and/or determining their identity, number, size or origin have long been in use. However, the staining of particles using optically detectable labels generally must be accompanied by one or more washing and/or centrifugation procedures to remove background interference from unbound label. Physical separation procedures, such as washing or centrifugation/ultracentrifugation, can lead to inefficiencies as well as inaccuracies in the analyses, especially when analyzing small volumes of sample, due to partial loss of particles during the separation.
In addition, the analyses of small particles, in the range of nanometers in diameter or less (e.g., about 100-200 nm or less), pose hurdles. For example, light scatter-based flow cytometry analyses of extracellular vesicles (EVs), exemplary of which are biological membrane vesicles that are released from cell surfaces (ectosomes), internal stores (exosomes) or as a result of apoptosis or cell death, often provide incorrect estimates of their size and concentration when the vesicles are nanovesicles, due to dim light scatter. Further, detection of the EVs often is triggered by coincidence, i.e., simultaneous detection of the presence of more than one EV in the flow cytometer measurement volume, leading to incorrect concentration, size and fluorescence estimates.
Some optical methods, such as nanoparticle tracking analysis (NTA), also are limited in their ability to measure nanoparticles, due to the particles scattering less light than the limits of detection. In addition, unlike flow cytometry, where the entire sample containing the particles passes through the measurement volume, particles can diffuse in and out of the probe volume during NTA measurements, resulting in over-counting of smaller particles and under-counting of larger particles. Improved optical methods are needed for the detection of particles, including nanoparticles, among which are EVs, which often are 500 nm or less in diameter.